The invention concerns instruments for fast, selective replication of deoxyribonucleic acid (DNA) from biomaterial by the well-known polymerase chain reaction (PCR), working in individual duplication thermocycles.
It is becoming more and more important for the medical care of patients that analysis methods in genetic engineering are made available which work very quickly. One example of this is the identification of infectious microorganisms, which still requires days at present, but actually requires treatment at the earliest possible stage, in the initial hours if possible. More intense will be the demand for quick analysis during examinations of tissue possibly affected by cancer or other disease during surgery on the open patient by means of oncogenetic, virological or bacteriological analyses. Here, a maximum analysis time of about ten minutes is required.
Mass spectrometry today provides very fast, highly sensitive analysis methods for the size of amplified DNA segments. Advances in matrix-assisted laser desorption and ionization (MALDI) make it possible to analyze about 20 samples including the MALDI preparation, the introduction of DNA MALDI samples into the mass spectrometer, the MALDI analysis and the data evaluation up to presentation on the screen in less than three minutes. The tissue cells and DNA extraction can be lysed in less than two minutes.
This maximum of five minutes total for sample preparation and mass spectrometry analysis stands in contrast to times of three hours for classic PCR replication. Extreme reductions in these times are on the horizon however. In one instrument, available commercially in the meantime, this time has already been reduced to about 20 minutes. In a recent publication (A. T. Woolley et al., xe2x80x9cFunctional Integration of PCR Amplification and Capillary Electrophoresis in a Microfabricated DNA Analysis Devicexe2x80x9d, Anal. Chem. 68, 4081, December 1996), DNA in 20 microliters of reaction solution was amplified through 30 cycles in only 15 minutes in a miniature chamber made of polypropylene. Even this time is, however, too long for a fast analysis in the above sense. The goal must be to perform the PCR amplification in only two to three minutes.
As is known, DNA consists of two complementary chains made up of four nucleotides, the sequence of which forms the genetic code. Each nucleotide consists of a sugar (ribose), a phosphoric acid group and a base. Two bases each are complementary to one another. Sugar and phosphoric acid form the continuous chain of the DNA (or the so-called backbone), the four characteristic bases are each lateral branches attached to the sugar. Both complementary chains or single strands of DNA are coiled around one another in the form of a double helix, whereby two complementary nucleotides each are connected to one another via hydrogen bridges between the bases and thus form a so-called double strand.
The basis for many analysis methods in genetics is the selectively functioning PCR (polymerase chain reaction), a simple replication method for specifically selected DNA pieces in a test tube, first developed in 1983 by K. B. Mullis (who received the Nobel Prize for this in 1993) and which, after the introduction of temperature stable polymerases, went on to unequalled success in genetic engineering laboratories.
PCR is the specific replication of a relatively short segment of double-stranded DNA, precisely sought from the genome, in simple temperature cycles. Selection of the DNA segment is through a so-called pair of primers, two DNA pieces with about 20 bases length apiece, which (described somewhat briefly and simply) encode the bilateral ends of the selected DNA segment. Replication is performed by an enzyme called polymerase, which represents a chemical factory in a molecule. The PCR reaction takes place in aqueous solution in which a few molecules of the original DNA and sufficient quantities of polymerase, primers, triphosphates of the four nucleic acids (so-called xe2x80x9csubstratesxe2x80x9d), activators and stabilizers are present. In every thermal cycle, the DNA double helix is first xe2x80x9cmeltedxe2x80x9d at about 95xc2x0 C., whereby both strands are separated from one another. At about 55xc2x0 C., the primers are then attached to complementary nucleotide sequences of the DNA single strands (xe2x80x9chybridizationxe2x80x9d). At 72xc2x0 C. the double helixes are reconstructed by elongation of the primers, done by the temperature-resistant polymerase (e.g. taq-polymerase). Complementary nucleotides are bonded, one after the other, to a specific end of the primers to form two new double helixes. In this way, the selected DNA segment is duplicated in principle between the primers. Therefore, over 30 cycles, around one billion DNA segments are generated from one single double-strand of DNA as original material. (In a more exact description, the shortening to the DNA segment between the primers only occurs statistically with further replications).
The duration of time for a thermal cycle is practically only dependent on the rate of heating up and cooling down, which is subsequently dependent upon the volume of liquid, the dimensions of the chamber and the thermal conductivity of the chamber walls and the reaction solution. For every thermal stage, only a few seconds are necessary in principle, sometimes even less.
In the above cited article by Woolley et al., in which the PCR amplification for 30 cycles only lasted 15 minutes, the following times were required, for example, for the work in the three thermal stages: 2 seconds at 96xc2x0 C. for melting, 5 seconds at 55xc2x0 C. for the primer attachment and 2 seconds at 72xc2x0 C. for reconstruction. The remaining time of 21 seconds per cycle was used for the thermal transitions. The DNA melts almost instantaneously at a temperature a few degrees above the xe2x80x9cmelting temperature.xe2x80x9d Analyses have shown that heating to this temperature for one half second suffices for complete separation of all double helix structures. Precise maintenance of the temperature is not even especially critical here, as long as one remains above the melting temperature but below a coagulation temperature. Hybridization also does not need much time if the primers are available in sufficient concentration. At an optimal concentration, about one to two seconds are enough. For hybridization, the temperature is even less critical; it need only remain under 60xc2x0 C. to proceed sufficiently fast. Optimal conditions are at about 55xc2x0 C.
The growth of the attached primers into a complementary DNA molecule through the polymerase, known as xe2x80x9creconstructionxe2x80x9d in the following, has a very high velocity. 500 to 1,000 bases can be bonded per second under optimal thermal and concentration conditions by the polymerase. Since generally only DNA segments of a maximum of 400 bases in length are necessary for the analyses, two seconds are quite sufficient for this reconstruction phase. For this process of reconstruction of a new double helix, good maintenance of the optimal temperature is required in order to achieve the high rate of reconstruction.
Theoretically, a PCR reaction cycle could thus be concluded in less than 5 seconds, under the precondition that heat can be introduced or removed up to each sufficient thermal equilibrium in about xc2xc second each. One such ideal thermal curve for a PCR cycle is shown in FIG. 1. The introduction and removal of heat are the critical time-determining variables here.
By the addition of only one primer pair, uniform DNA segments can be replicated. However, if several different primer pairs are added at the same time, several DNA segments will also be replicated at the same time (xe2x80x9cmultiplexed PCRxe2x80x9d). This type of multiplexed PCR is frequently used and often has special advantages. For so-called xe2x80x9cfingerprintingxe2x80x9d for the identification of individuals through DNA segments of variable length (methods of xe2x80x9cVNTR=Variable Number of Tandem Repeatsxe2x80x9d or xe2x80x9cAMPxe2x88x92FLP=Amplified Fragment Length Polymorphismxe2x80x9d), it makes the analyses faster. Here through the selection of primers, which determines the average molecular weight of the DNA segments, the result can be achieved that the variations of molecular weights for the DNA segments formed by the various primer pairs only seldomly or never overlap. This type of multiplexed PCR requires an analyzer which is capable of simultaneous measurement of a large range of molecular weights. The method is particularly advantageous for the identification of infectious organisms, since 20 types of bacteria (or viruses, yeasts, molds) can be detected at the same time, for example, with a single PCR replication.
The high sensitivity of modern measurement methods for the analysis of DNA, for example the sensitivity of the above-mentioned mass spectrometric measurements, permits the volume of reaction solution to be reduced while maintaining the optimal concentration. Since on the one hand, for the same initial amount of DNA, the reaction solution is then exhausted after a few cycles (though on the other hand not very much amplified DNA material is required for the analysis) the number of cycles can be reduced from the normal amount of 30 to about 24 to 28. However, the time-saving due to this is minimal. Possible reduction of the volumes suggests a solution based on microfabrication technologies for a new PCR amplification method such as has already been applied in the above cited article by Woolley et al.
Also in the review article xe2x80x9cMicrofabrication Technologies for Integrated Nucleic Acidxe2x80x9d, D. T. Burke, M. A. Burns and C. Mastrangelo, Genome Research 7, 189 (1997), chambers manufactured using microfabrication technology are presented for PCR amplification, without however giving any indication of the achievable rates. Such chambers, 1,000xc3x971,000xc3x97250 micrometers large here and made of a low temperature polymer, nevertheless have the disadvantage that they can only be emptied by extended rinsing with a washing liquid and thus force a dilution of the amplified DNA when emptying.
Another obvious idea is to allow the reaction solution to run constantly through a fine capillary which crosses three zones, kept stationarily at the appropriate temperatures, on a microfabricated chip in a simple manner for every cycle, whereby the standard temporal variation in the temperature is replaced by a simple local variation in temperature. A section of one such arrangement is shown in FIG. 3. A small dimension for the capillary should then allow a rapid temperature change up to thermal equilibrium.
Unfortunately, the flow in a capillary impairs the work of the polymerase in the reconstruction phase. In a cylindrical capillary, a laminar flow with a parabolic velocity profile generally prevails, whereby the average velocity is doubled in the central axis of the capillary while it is zero at the margin of the capillary. In a capillary with a square or rectangular cross section, somewhat different conditions prevail, however the differences are not decisive here. The flowing reaction solution is therefore divided into sliding layers of differing velocity, while adjacent molecules in different sliding layers move past one another. The individual molecules are subject to shearing forces. Straight molecules are aligned parallel to the direction of flow. For a close-to-real average velocity of 2 millimeters per second in a capillary 100 micrometers in diameter, two almost spherical molecules which are in contact with one another on both sides of an imaginary sliding surface, move past one another in one millisecond by about 8% of their diameter on average. One millisecond corresponds to the minimum time for the incorporation of a base. Molecules in the center of the flow do not experience this sort of displacement. Molecules close to the wall of the capillary experience a greater displacement. In this way, however, the work of the polymerase which requires a calm, adjacent positioning of the molecules on a millisecond scale, is greatly impaired. Increased errors are the result and, with even greater displacement motion, the polymerase work is even stopped. The displacement motion of adjacent molecules increases for the same flow in proportion to the third power of the reciprocal diameter of the capillary. There is therefore a dilemma for the flow PCR: thinner capillaries improve the temperature setting, however they extend the distance, therefore necessitating an increased flow rate and thus impairing amplification.
The invention makes use of extremely brief cycle times of only a few seconds for the PCR reactions. These reaction are generated by using PCR reaction chambers constructed as a pattern of fine capillaries in close proximity to heating and cooling elements, and by keeping the flow rates in the capillaries to a minimum during the amplification phase so that the polymerase reaction is not disturbed. In this way, the temperature cycles in the reaction solution for the three temperature phases of the PCR duplication can be optimally shortened in duration. The capillary pattern can be simply produced by means of microfabrication technology.
It is the basic idea of the invention to use, on the one hand, a pattern of very fine capillaries in close proximity to heating and cooling elements as a chamber system for the reaction solution in order to keep the heating and cooling-down times for the reaction solution extremely low, while on the other hand however keeping the flow rate for the reaction solution in the capillaries during the reconstruction phase of the DNA double strand using the polymerase as low as possible. The flow rate during the reconstruction phase should never exceed ten times the maximum capillary diameter prevalent there per second, while more favorable would be a medium flow rate of less than five maximum capillary diameters per second. The error rate for the reconstruction only approaches its minimum below a medium flow rate which is less than double the diameter per second. The maximum capillary diameter corresponds to the normal diameter for round capillaries, for rectangular cross sections that of the diagonal.
A favorable, very fine capillary structure with closely adjacent heating elements may be favorably produced using microfabrication technologies. The low flow rate can be provided on the one hand (especially at a constant flow of reaction solution through the capillary structure) by a special design of the capillary net, on the other hand, the low flow rate may also be produced by special methods of application with temporally changeable flows of the reaction solution.
The advantage of a fine capillary structure is evident: the times for the thermal transitions in the reaction solution may be kept very short. This advantage is however opposed by severe disadvantages: the extremely large surface area of the chamber system disturbs the biochemical processes if the surface even only minimally influences the affected molecules. Thus for example a bare silicon surface immediately kills the activity of the polymerase. Many plastics too have proven to be unsuitable for the PCR. Even the same plastics from different manufacturers, for example the normally favorable plastics polyethylene or polypropylene, have had different types of effects on the PCR due to their varying qualities. Therefore, the surface must very thoroughly be made completely inert.
The activity of a surface can be almost completely eliminated by a thorough coating. Coating methods for capillaries are known from chromatography, especially from gas chromatography, which eliminate even the smallest remnant of active surface. Particularly coatings with thread-shaped molecules which are bonded monolaterally onto the surface (xe2x80x9cchemically bonded phasesxe2x80x9d), have generated thermally stable and extremely inert surface coatings. Here, hydrophobic or hydrophilic, polar or nonpolar, fat or water absorbent surface coatings can be generated which may also have other characteristics within the depth of the layer. It is therefore a further idea of the invention to use the known chromatographic coatings for the deactivation of surfaces. Particularly for the coating of quartz glass and glass surfaces on the interior of thin capillaries, explicit and comprehensive formulas with descriptions of the necessary steps are available. Silicon surfaces can be transformed by oxidation into quartz surfaces.
Particularly for metal implants, stable coatings have been developed which correspond to endogenous proteins and glycoproteins such as occur in cell membranes. Such coatings may reduce the activities on the surfaces for polymerase reactions in the present case, even if they are not yet successful as implant coatings. The micromanufacturing methods, however, also comprise the molding of plastics in micromanufactured silicon forms. In this way as well, capillary systems can be developed which may be used as reaction chambers. It is therefore a further idea of the invention to use favorable polymers such as low pressure polyethylene or polypropylene for the manufacture of capillary systems. Since polymers normally possess poor thermal conductivity characteristics, these may also be filled with thermally well conducting nanopowders, for example with silver powder. These powders can be produced with a particle diameter of about 10 to 1,000 nanometers. They are excellently suited for increasing the thermal conductivity of plastics. The powders may be deposited in such a way that they do not directly lie on the surface. The low flow rate necessary for this invention can be achieved in a constantly circulating capillary system, whereby zones of different temperatures are passed through, in such a way that the flow of the reaction solution in the zone of reconstruction temperature branches off into a multitude of parallel capillaries, in which the flow rate in each of these parallel capillaries is reduced as shown in FIG. 4.
The reaction solution can also be moved on intermittently by pressure pulses. After each filling of the capillary system for the reconstruction of the DNA double strand, at the corresponding temperature, the flow of the reaction solution stops, the incorporation reactions run down and only then (after about 2 seconds) is the reaction solution pumped on. It is therefore advantageous to keep each of the volumes at equal amounts for the chamber systems for melting, attachment of primers, and reconstruction, so that the reaction solution is always pressed on by exactly this amount of volume. A pulsed process occurs which, however, makes it imperative for the dwell times of the reaction solution to be equal in the three temperature zones.
It is however also possible, in particular, to select a capillary system large enough so that the entire quantity of reaction solution to be processed can be held in it and then very quickly passed through the temperature phases one after another using fast heating and cooling elements with the solution at rest.
Such a type of capillary system may easily be aligned in one plane, as shown in FIGS. 2a and 2b. The capillaries arranged in a plane are enclosed in a thin membrane, on the surface of which there are heating elements, also in a planar structure. Thus for example, 200 nanoliters of reaction solution in 16 parallel capillaries with cross sections of 60xc3x97100 micrometers and 2 millimeters length can be located on a surface of about 2xc3x971.6 millimeters. These capillaries are located in a silicon membrane of 300 micrometers maximum thickness. Through the thin membrane and through the bridges between the capillaries, heat can be applied or discharged very efficiently.
On the top and bottom of the membrane, there are resistance grids planarly imbedded or otherwise attached, which take care of the heating capacity. With less than two watts heating capacity, the temperature of this type of thin silicon membrane with a surface of 3xc3x973 mm2 can be raised by about 100xc2x0 C. per second, an increase from 45xc2x0 C. to about 72xc2x0 C. can therefore be achieved in 0.3 seconds. The temperature can itself be determined in the known fashion via the thermal coefficient from the resistance of the heating element. Control of the heating capacity with a slight overshoot leads to quick adjustment of the equilibrium in the reaction solution. Via gaseous, liquid or solid movable cooling means, which can be brought into planar contact with the membrane the membrane can be cooled very quickly. An arrangement with a solid cooling element is depicted in FIG. 2b. In the simplest case, the cooling means may be at room temperature, or at a lower temperature for acceleration. Since the temperature for primer attachment need not be exactly adjusted, a simple time control is sufficient for the contact time. In more critical cases, the change in resistance for the heating elements may be exploited as a control of the contact time. The cooling means, moved for example electromechanically or pneumatically, may be a part of the microsystem arrangement, or they may also be brought in contact with the membrane through external movement devices.